www.nature.com/scientificreports OPEN Whole exome sequencing identifed sixty-fve coding mutations in four neuroblastoma tumors Received: 30 September 2016 Aubrey L. Miller1, Patrick L. Garcia1, Joseph G. Pressey2,8, Elizabeth A. Beierle3, David R. Accepted: 20 November 2017 Kelly4,5, David K. Crossman6, Leona N. Council4,7, Richard Daniel5, Raymond G. Watts2,9, Published: xx xx xxxx Stuart L. Cramer2,10 & Karina J. Yoon1 Neuroblastoma is a pediatric tumor characterized by histologic heterogeneity, and accounts for ~15% of childhood deaths from cancer. The fve-year survival for patients with high-risk stage 4 disease has not improved in two decades. We used whole exome sequencing (WES) to identify mutations present in three independent high-risk stage 4 neuroblastoma tumors (COA/UAB-3, COA/UAB -6 and COA/ UAB -8) and a stage 3 tumor (COA/UAB-14). Among the four tumors WES analysis identifed forty- three mutations that had not been reported previously, one of which was present in two of the four tumors. WES analysis also corroborated twenty-two mutations that were reported previously. No single mutation occurred in all four tumors or in all stage 4 tumors. Three of the four tumors harbored genes with CADD scores ≥20, indicative of mutations associated with human pathologies. The average depth of coverage ranged from 39.68 to 90.27, with >99% sequences mapping to the genome. In summary, WES identifed sixty-fve coding mutations including forty-three mutations not reported previously in primary neuroblastoma tumors. The three stage 4 tumors contained mutations in genes encoding protein products that regulate immune function or cell adhesion and tumor cell metastasis. Neuroblastoma (NB) is an embryonal tumor arising from neural crest cells of the sympathetic nervous system1. It is the most common extracranial solid tumor of children, and accounts for ~15% of all childhood cancer deaths. Treatment of children with high-risk disease has been a major challenge in pediatric oncology. Patients less than 18 months of age with low risk disease attain cancer-free status with tumor resection alone or without interven- tion, due to spontaneous tumor regression2. In contrast, patients older than 18 months of age who have high-risk factors such as MYCN amplifcation, bilateral disease, and near-diploid or near-tetraploid karyotype ofen relapse afer initial treatment and remission, with an almost uniformly fatal outcome3–6. Te new International Neuroblastoma Risk Group (INRG) Staging System has taken advantage of recent advances in medical imaging and biomolecular diagnostics to establish a consensus for risk stratification5. Te criteria for classifcation include stage, age, histology, tumor grade and MYCN gene copy number. Criteria for high-risk NB include age greater than 18 months, stage 2 or 3 with MYCN amplifcation, and unfavorable histology6. Genetic abnormalities associated with high-risk stage 4 NB include hemizygous deletions of the q arm of chromosome 11 (up to 62.5% of tumors) and of the p arm of chromosome 1 (25–35% of tumors), and MYCN amplifcation in ~25% of tumors3,4,7–12. Gains in the long arm of chromosome 17 (17q21–17qter) is one of the most frequent genetic alterations in NB, occurring 50–70% of all high-risk tumors3,4. Recent advances in next-generation sequencing technology and a collaboration between The Pediatric Tumor Bank and Tumorgraf Development Initiative at Children’s of Alabama and the University of Alabama 1Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA. 2Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, USA. 3Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA. 4Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA. 5Department of Pathology and Laboratory Medicine, Children’s of Alabama, Birmingham, AL, USA. 6Department of Genetics, University of Alabama at Birmingham, Birmingham, AL, USA. 7The Birmingham Veterans Administration Medical Center, Birmingham, AL, USA. 8Present address: Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA. 9Present address: Department of Pediatrics, LSUHSC School of Medicine, New Orleans, LA, USA. 10Present address: Palmetto Health Children’s Hospital, Columbia, SC, USA. Correspondence and requests for materials should be addressed to K.J.Y. (email: [email protected]) SCIENTIFIC REPORTS | 7: 17787 | DOI:10.1038/s41598-017-17162-y 1 www.nature.com/scientificreports/ Tumor INRG* Diferentiation MYCN >18 Tumor ID Type Stage Staging (Grade) amplifed months COA/ UAB-3 NB 4 M Poor Yes Yes COA/ UAB-6 NB 4 M Poor Yes Yes COA/ UAB-8 NB 4 M Poor No Yes COA/ UAB-14 NB 3 L2 Poor No No Table 1. Clinical characteristics associated with four primary neuroblastoma tumors. *INRG: International Neuroblastoma Risk Group. COA/UAB-3 COA/UAB-6 COA/UAB-8 COA/UAB-14 Not Not Not Not Variants Types reported Reported reported Reported reported Reported reported Reported Nonsynonymous coding1 12 3 6 7 4 1 13 8 Nonsynonymous start2 Splice site acceptor3 1 1 1 Splice site donor3 Start gained4 2 1 1 1 Start lost4 1 Stop gained5 2 1 Stop lost5 TOATL # VARIANTS 16 3 7 8 5 2 16 9 TOTAL # GENES 15 3 7 8 4 2 15 9 Table 2. Summary of variants (mutations) types for all mutations identifed in four neuroblastoma tumors. 1Mutation of a single nucleotide, resulting in an amino acid change in the encoded protein; may afect phenotype66. 2Mutation that occurs in a coding region, at start site. 3Mutation that changes nucleotides in genomic loci where splicing takes place. 4Mutation that generates a new translation initiation codon in the 5′UTR, or that results in the loss of an initiation codon. Start site loss may result in the loss of protein product. 5Mutation that changes the sequence of a codon to create or remove a stop codon (UAA, UAG, UGA). at Birmingham (COA-UAB) facilitated performing whole exome sequencing (WES) to analyze four recently acquired neuroblastoma specimens. Te goals of the study were to sequence the exome of these primary tumors using Whole Exome sequencing to identify mutations, to generate CADD (Combined Annotation Dependent Depletion) scores as a measure of predicted pathogenicity of mutated gene products, and to compare WES data of the stage 3 tumor with the three stage 4 tumors. Results Clinical characteristics associated with primary neuroblastoma tumors in this study. Primary tumors were received from patients who underwent surgery as standard of care at Children’s of Alabama Hospital (Table 1). Tumors were obtained from patients diagnosed with intermediate (COA/UAB-14) or high-risk dis- ease (COA/UAB-3, COA/UAB-6, COA/UAB-8). Tumors COA/UAB-3 and COA/UAB-6 were MYCN amplifed. Tumor specimens COA/UAB-3, /UAB-6, and /UAB-8 were obtained from patients older than 18 months, and had high-risk characteristics that included unfavorable histology and MYCN amplifcation. WES identifed 43 mutations not reported previously in four neuroblastoma tumors. WES analysis revealed that each tumor harbored between seven and twenty-fve mutations (Table 2). Te average of 16 mutations per tumor is consistent with previous reports of 12–18 mutations per tumor13,14. Te four tumors harbored 43 mutations not previously observed in NB tumors in the dbSNP database (version 138), as well as 22 mutations reported previously to be present in other tumor types15. Tose 43 mutations are in bold in Tables 2–6. In Tables 3–6, ‘p’ in the third column of each Table identifes the amino acid substitution and position; ‘c’ in this column identifes the nucleotide substitution and position. While no mutation was common to all four tumors, one of the mutations in the RHPN2 gene was present in two of the four tumors examined: the mutation in this gene (Rhophilin, Rho GTPase Binding Protein 2) was present at nucleotide 217 (G > A encoding Val73Met) in COA/UAB-3 and COA/UAB-8 tumors (Tables 3–5). RHPN2 contributes to actin cytoskeleton organization, an organelle that regulates cell motility16,17. A second mutation introducing a start site of RHPN2 gene also occurred in tumors COA/UAB-3 and COA/UAB-8. Te location of the introduced start site at the intron-exon bound- ary suggests that this mutation is unlikely to alter the protein product in tumors COA/UAB-3 and COA/UAB- 8. A genome-wide association study (GWAS) found that a region containing RHPN2 has been associated with increased susceptibility to colorectal cancer18. Genes encoding MUC4 and ADAM21 also contained mutations in two of the four tumors, but at diferent loci. Mucin 4 (MUC4), a transmembrane mucin expressed predominantly by normal epithelial cells, is involved in cell diferentiation, inhibition of cell adhesion, and cell migration19–21. MUC4 protein is thought to contribute SCIENTIFIC REPORTS | 7: 17787 | DOI:10.1038/s41598-017-17162-y 2 www.nature.com/scientificreports/ Mutation Ch#+ Gene Mutation type Known functions/pathways of normal gene product 1* TCEB3 p.Ala18Val/c.53 C > T Missense Activates RNA polymerase II elongation 1* TOE1 p.Ala2Val/c.5 C > T Missense Inhibits cell growth and cell cycle progression 1 MAEL p.Tyr344Asn/c.1030 T > A Missense Spermatogenesis 1 SELL Start gained Mediates adhesion 2* WDR35 p.Ala1018Asp/c.3053 C > A Missense Promotes CASP3 activation 2* COL4A4 p.Gly645*/c.1933G > T Nonsense Major structural component of basement membrane 3 MUC4 p.Ala1646Tr/c.4936 G > A Missense Plays a role in tumor progression; anti-adhesive properties 6 CLIC5 p.Gln50His/c.150 G > T Missense Chloride ion transport 6 FOXO3 p.Glu17Val/c.50 A > T Missense Apoptosis; transcriptional activator 13 ITM2B p.Ala153Val/c.458 C > T Missense Processing beta-amyloids A4 precursor protein (APP) 14 RNASE4 p.Cys85Phe/c.254 G > T Missense Degrades RNA 14 ADAM21 p.Pro40Leu/c.119 C > T Missense Adhesion protein involved in sperm maturation; epithelial cell function 17 ACADVL p.Phe266Leu/c.798 C > A Missense Mitochondrial fatty acid beta-oxidation 19 GIPR p.His115Asn/c.343 C > A Missense Pathogenesis of diabetes Binds to and activates GTP-Rho, negatively regulates stress fber 19 RHPN2 p.Val73Met/c.217 G > A Missense formation and facilitates motility of many cell types including T and B cells.